Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysine residues in histone amino termini, leading to chromatin condensation and changes in gene expression. Reversible lysine acetylation is an important phenomenon for homeostatic regulation of many cellular processes. The best characterized proteins that are subjected to this mode of regulation are histones (Strahl, B. D et al., Nature 2000, 403, (6765), 41-5). Lysine residues in the N-terminal tail are tightly regulated by acetylation and deacetylation catalyzed by enzymes known as histone acetyltransferase (HAT) or histone deacetylase (HDAC) (Minucci, S. et al., Nat Rev Cancer 2006, 6, (1), 38-51; Yang, X. J. et al., Oncogene 2007, 26, (37), 5310-8). Acetylation of lysines in the histone H3 and histone H4 tails is strongly correlated to chromatin states that are primed for transcription, or that are part of actively transcribed genomic regions (Strahl, B. D. et al., Nature 2000, 403, (6765), 41-5; Minucci, S. et al., Nat Rev Cancer 2006, 6, (1), 38-51). Acetylation of histones has also been correlated with other important cellular functions including chromatin assembly, DNA repair, and recombination.
There are 18 HDAC enzymes in the human genome that are subdivided into four distinct classes (Lane, A. A. et al., J Clin Oncol 2009, 27, (32), 5459-68; Marks, P. et al., Nat Rev Cancer 2001, 1, (3), 194-202). Classes I, II and IV (11 enzymes) contain a zinc (Zn2+) molecule in their active site. Class III contains seven mechanistically diverse NAD+-dependent enzymes known as sirtuins. Class II is subdivided into Class IIa (HDAC4, 5, 7 and 9) and Class IIb (HDAC6 and HDAC10).
Alterations in histone modifications have emerged as one of the key mechanisms responsible for tumor transformation (Conley, B. A. et al., Cancer 2006, 107, (4), 832-40; Glozak, M. A. et al., Oncogene 2007, 26, (37), 5420-32). Altered expression and mutations of genes that encode HDACs have been linked to tumor development since they both induce the aberrant transcription of key genes regulating important cellular functions such as cell proliferation, cell-cycle regulation and apoptosis (Lane, A. A. et al., J Clin Oncol 2009, 27, (32), 5459-68; Marks, P. et al., Nat Rev Cancer 2001, 1, (3), 194-202). Hence inhibitors of histone deacetylase enzymes (HDACi) have recently attracted substantial attention as potential anti-cancer drugs. The clinical relevance of this attention that is warranted has recently been underscored by the introduction of vorinostat (Zolinza™, Merck, also widely known SAHA=suberoylanilide hydroxamic acid), Romidepsin (Istodax) and Belinostat for the treatment of cutaneous T-cell lymphoma (Marks, P. A., et al., Expert Opin Investig Drugs 2010, 19, (9), 1049-66).
Class II HDAC enzymes exhibit tissue-specific expression and can shuttle between the nucleus and cytoplasm. There is a growing interest in this class of HDAC enzymes because their substrates are broader and not limited to histones. For example, Class IIb enzyme HDAC6 predominantly resides in cytoplasm and hence its substrates are nonhistone proteins including α-tubulin, cortactin, peroxiredoxins, chaperone proteins, HSP90, β-Catenin, hypoxia inducible factor-1α (HIF-1α) and other proteins (Li, Y. et al., FEBS J 2013, 280, (3), 775-93; Shankar, S. et al., Adv Exp Med Biol 2008, 615, 261-98).
HDAC6 contains two functional homologous catalytic domains and an ubiquitin-binding zinc finger domain at the C-terminal region. HDAC6 is an authentic protein lysine deacetylase and appears to be important for a myriad biological processes and aberrant regulation of HDAC6 is implicated in numerous pathological conditions from cancer to neurodegenerative diseases (Valenzuela-Fernandez, A. et al., Trends Cell Biol 2008, 18, (6), 291-7; Simoes-Pires, C. et al., Mol Neurodegener 2013, 8, (1), 7).
HDAC6 stably associates with tubulin and regulate its acetylation states. Since microtubules are at the heart of cellular self-organization, it is not surprising that the deacetylation activity of HDAC6 towards tubulin affects many cellular processes. HDAC6 is known to play important roles in cell migration and cell-cell interaction. Aberrant regulation of HDAC6 is associated with cancer development (Valenzuela-Fernandez, A. et al., Trends Cell Biol 2008, 18, (6), 291-7; Simoes-Pires, C. et al., Mol Neurodegener 2013, 8, (1), 7). For example, overexpression of HDAC6 correlates with invasive metastatic behavior of tumor cells (Aldana-Masangkay, G. I. et al., J Biomed Biotechnol 2011, 875824). Moreover, HDAC6 directly or indirectly regulates angiogenesis by deacetylating several key factors that control angiogenesis (Li, Y. et al., FEBS J 2013, 280, (3), 775-93; Aldana-Masangkay, G. I. et al., J Biomed Biotechnol 2011, 875824). Recent studies also suggest HDAC6 regulates acetylation of beta-catenin in CD133 signaling pathway which is known to be important for tumor stem cell maintenance (Mak, A. B. et al., Cell Rep 2012, 2, (4), 951-63).
HDAC6 has also been linked to cell survival pathways through several different mechanisms. HDAC6 regulates reversible acetylation of Hsp90 chaperon whose client proteins include steroid hormone receptors and a number of protein kinases critical for cell proliferation and apoptosis. Inactivation of HDAC6 perturbs the chaperon activity of Hsp90 and attenuates the activity of growth promoting client proteins (Aldana-Masangkay, G. I. et al., J Biomed Biotechnol 2011, 875824). Through its ubiquitin binding domain, HDAC6 can bind polyubiquitinated misfolded proteins and deliver them to the dynein motor proteins for transport into aggresomes for degradation by lysosomes (Kawaguchi, Y. et al., Cell 2003, 115, (6), 727-38). HDAC6 also plays a role in the eventual clearance of aggresomes by promoting fusion of autophagosome with lysosomes (Lee, J. Y. et al., EMBO J 2010, 29, (5), 969-80; Iwata, A. et al., J Biol Chem 2005, 280, (48), 40282-92; Pandey, U. B. et al., Nature 2007, 447, (7146), 859-63).
Selective inhibition of HDAC6 can enhance apoptotic response to DNA damaging agents such as etoposide and doxorubicin (Namdar, M. et al., Proc Natl Acad Sci USA 2010, 107, (46), 20003-8). Conversely there is also evidence supporting a role of inhibition of HDAC6 in protecting normal cells from DNA-damage induced cell death and promote neuron regeneration (Rivieccio, M. A. et al., Proc Natl Acad Sci USA 2009, 106, (46), 19599-604). Thus, inhibition of HDAC6 may dramatically improve therapeutic index of cytotoxic agents.
HDAC6 is a target for protection and regeneration following injury in the nervous system. Injury of neurons leads to an increase in HDAC6 expression and inhibition of HDAC6 can promote survival and regeneration of neurons. Importantly, selective inhibition of HDAC6 avoids cell death associated with non-selective HDAC inhibitors (pan-HDAC inhibitors). Therefore HDAC6 may be promising target for the treatment of, for example, stroke, ischemia and spinal cord injury (Rivieccio, M. A. et al., Proc Natl Acad Sci USA 2009, 106, (46), 19599-604).
It is advantageous to have a selective HDAC6 inhibitor that inhibits HDAC6 with greater potency than other HDACs with no unwanted side effects (Bradner et al., Nat. Chem. Biol., 2010, 6(3):238-243; WO-A2011/019393).
In view of the importance of inhibiting only those HDAC isoforms relevant to a disease state, minimizing acetylation of proteins not related to the disease, and reducing side effects and toxicity, new HDAC inhibitors that are selective for specific HDACs are needed.
TTK/Mps1, a dual specificity protein kinase, has emerged as a master regulator of mitosis. In agreement with its proposed function in highly proliferative cells, elevated level of TTK/Mps1 is found in a variety of human cancer cell lines and primary tumor tissues. Like many cell cycle regulators, Mps1 transcription is deregulated in a variety of human tumors. Elevated Mps1 mRNA levels are found in several human cancers, including thyroid papillary carcinoma, breast cancer, gastric cancer tissue, bronchogenic carcinoma, and lung cancers (Mills, 1992 #187; Salvatore, 2007 #209; Yuan, 2006 #216; Kilpinen, 2010 #197; Daniel, 2010 #49; Landi, 2008 #217). Furthermore, high levels of Mps1 correlate with high histological grade in breast cancers (Daniel, 2010 #49). Conversely, Mps1 mRNA is markedly reduced or absent in resting cells and in tissues with a low proliferative index (Hogg, 1994 #190). Thus, there is a correlation between elevated Mps1 levels and cell proliferation as well as tumor aggressiveness. Consistent with the notion that oncogenic signaling promotes Mps1 expression, the levels and activity of Mps1 are increased by 3 and 10 fold respectively in human melanoma cell lines containing B-Raf (V600E) mutant (Cui, 2008 #153). Inhibition of B-Raf or MEK1 reduces Mps1 expression (Borysova, 2008 #147; Cui, 2008 #153).
The observation that tumor cells frequently over express spindle checkpoint proteins is perplexing as the conventional wisdom would postulate that tumor cells would have a weakened checkpoint, contributing to chromosome mis-segregation and aneuploidy. Indeed, significant evidence from yeast to mice supports the notion that a weakened checkpoint leads to chromosome instability (Weaver, 2005 #218). However, mutations in key checkpoint proteins are rare in human tumors, and correlative evidence showing that compromised checkpoint signaling directly contributes to the development of human tumors has been elusive. MPS1 missense mutations have been found in the noncatalytic, N-terminus in bladder (Olesen, 2001 #380) and lung cancers (Nakagawa, 2008 #406), and in the kinase domain in pancreatic (Carter, 2010 #384) and lung cancers (Nakagawa, 2008 #406). Interestingly, frameshift mutations that truncate the protein arise from microsatellite instability in the hMps1 gene in gastric (Ahn, 2009 #383) and colorectal cancers (Niittymaki, 2011 #382). Thus, mutations in hMPS1 have been detected in tumor-derived cells; however, their influence on tumorigenesis is not known.
The prevalence of high levels of checkpoint protein expression, such as Mps1, in human tumors prompts an alternative hypothesis regarding the potential role of checkpoint proteins in cancer cells, i.e. overexpression of these proteins may promote either cancer initiation or survival of aneuploid cancer cells (Sotillo, 2007 #219; Daniel, 2010 #49). Accordingly, reductions in key checkpoint proteins should severely decrease human cancer cell viability. This prediction is confirmed for several checkpoint proteins, including Mps1 (Fisk, 2003 #118; Daniel, 2010 #49), BubRI (Janssen, 2009 #173) and Mad2 (Kops, 2004 #220; Michel, 2004 #221). Suppression of Mps1 expression in Hs578T breast cancer cells also reduces the tumorigenicity of these cells in xenografts. The cancer cell death is likely due to severe chromosome segregation errors when the checkpoint is disabled. Interestingly, cells that survived reduced Mps1 levels often display lower levels of aneuploidy, suggesting that lower levels of Mps1 potentially inactivating the checkpoint are incompatible with aneuploidy (Daniel, 2010 #49). This concept is in excellent agreement with the observation that reduction in checkpoint proteins makes tumor cells more sensitive than untransformed human fibroblast to low doses of spindle poisons (Janssen, 2009 #173). Differential cellular responses to checkpoint inhibition between normal and tumor cells could be key in developing potential new anticancer drugs targeting hMps1.
Since different tumors have different levels of TTK expression, cancers that are most likely benefit from anti-TTK therapy are those tumors that express very high levels of TTK/Mps1. There is a need for effective methods for identification of cancerous cells by detection of expression levels of TTK/Mps1 in tumor biopsy. Reinhard et al from Chrion Corporation filed a US patent in 2005 (US 20050058627) claims TTK can be used a tumor diagnostic marker and as a therapeutic target for cancer therapeutics.
Several TTK/Mps1 inhibitors have been described in the literature and patents. This list includes SP600125 (IC50=250 nM), 2-Anilinopurin-8-ONES (AZ3146, IC50=35 nM), Mps1-IN-1 (IC50=370 nM), reversine (IC50=3 nM), NMS-P715 (IC50=8 nM) and MPI-0479605 (IC50=3.5 nM). NMS-P715 has been tested in an ovarian xenograft and reported promising efficacies. Overall the TTK/Mps1 inhibitor development is still in a very early preclinical stage. Despite of availability of different small molecule chemotypes of Mps1 inhibitors (Reviewed in Liu and Winey, Annual Review of Biochemistry 2012), a fundamental question that has been addressed is that whether Mps1 inhibitors as singular agent can be effective in cancer therapeutics. First of all the therapeutic index of Mps1 inhibitor is rather narrow which is consistent with the essential function of Mps1 in both normal and cancer cell proliferation. Consistent with this notion, animal xenograft studies clearly indicates that Mps1 inhibition exhibited significant neutropenia and animal toxicity (body weight loss and death) (Brandi Williams, Molecular Cancer Therapeutics Paper 2011, Mol Cancer Ther. 2011 December; 10(12):2267-75. doi: 10.1158/1535-7163.MCT-11-0453. Epub 2011 Oct. 6). The current studies clearly revealed that using Mps1 inhibitor as singular agent clearly has its limitation in cancer therapeutics. New concepts, methodology and target agents are sorely needed to overcome these barriers to successful cancer therapeutics.
During the inventors' investigation of Mps1 biology, they discovered that histone deacetylase inhibitors (HDACi) have unexpected regulatory effects on Mps1 function. Specifically they discovered that HDAC inhibitors increase the therapeutic index of Mps1 inhibitors and hypothesized that HDAC inhibitors prevent normal cells but not cancer cells to enter mitosis. In doing so the effects of Mps1 inhibition will only manifest in tumor cells as normal cells stall prior to entrant into mitosis in the presence of HDACi. Another mechanism is that HDAC6 inhibition perturbs the pathway that is essential for Mps1 kinase activation. It is well established that HDAC6-HSP90 signaling axis is required for maturation of active Mps1. Inhibition of HDAC6 exacerbates the effects of Mps1 inhibitor. Here we demonstrate that combination of an HDAC inhibitor with a Mps1 inhibitor results in robust tumor inhibition and minimal cytotoxicity. In addition, dual inhibitors that combine HDAC inhibitory activity with Mps1 inhibitory active is highly effective in tumor growth inhibition in vivo.
The present invention describes new selective inhibitors of HDAC6 and/or TTK/Mps1 Kinase.